PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a severe and prevalent inherited cardiac defect, characterized by ventricular chamber enlargement and systolic dysfunction. Although DCM is commonly associated with mutations in genes associated with contractility and other myocyte-specific functions, fibrotic and endothelial dysfunctions in patients suggest non-myocytes can influence disease pathogenesis and progression. Cardiac myocytes actively secrete a diverse array of proteins and vesicles into the extracellular milieu, the contents of which can change dynamically in response to stress and disease, suggesting a potential avenue of crosstalk communicating disease status between myocytes and non-myocytes. Thus far, our understanding of the cardiac secretomes is incomplete, hampered by difficulty of differentiating proteins secreted by the heart vs. other organs in patient plasma. To overcome this challenge, we propose to leverage cutting-edge iPSC technology, genome-editing technology, and proteomics technology to discover and validate cardiac secretomes and the crosstalk signaling pathways they regulate in the context of DCM pathogenesis. To identify the complement of secreted proteins from healthy and diseased cardiac cells, we first propose to generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from DCM patients with three common sarcomeric mutations. To clarify the detailed molecular mechanisms, we will conduct structural, electrophysiological, developmental, transcriptomic, and mechanistic analyses using patient- specific as well as genome-edited isogenic iPSC-CMs. This isogenic human iPSC platform will then be used to systematically discover the (i) secreted proteins and (ii) secreted exosomes of cardiac cells using large- scale proteomics platforms capable of quantifying hundreds of low-abundance proteins of interest. To confirm the signaling modality of secreted proteins, we will perform detailed transcriptomic and functional analysis of iPSC-derived endothelial cells (iPSC-ECs) and iPSC-derived cardiac fibroblasts (iPSC-CFs) co-cultured with diseased vs. healthy iPSC-CMs using high-throughput platforms. We anticipate that the successful completion of these studies will lead to new mechanistic insights into DCM pathogenesis, and help identify novel therapeutic targets that can impede and revert disease crosstalk signaling between myocytes and non- myocytes in the diseased heart. In a Supplement to the Parent R01 HL141371, we propose to leverage patient-derived human induced pluripotent stem cell (iPSC) platform towards studying mechanisms of CHD in people with Down syndrome. We hypothesize overexpression of cardiac-specific, dosage-sensitive trisomy genes on chromosome 21 leads to heart defects through impaired cardiac crosstalk and myocyte maturation. Aim 1 will generate a biorepository of 40 Down syndromes-pecific iPSC lines. To investigate the role of intercellular crosstalk in the pathogenesis of Down syndrome-related CHD, we will engineer iPSC-cardiac organoids resembling the heart tissue composition of cardiomyocytes, endothelial cells, and fibroblasts and determine molecular and functional phenotypes of cardiac organoids derived from the Down syndrome iPSCs. In Aim 2, we will investigate the mechanism of Down syndrome-related CHD using a pan-omic approach. The mechanisms of identified gene candidates will be further investigated through genome editing strategy. Completing the aims of this supplement will likely increase our understanding of Down syndrome-related CHD as well as broaden the overall impact of the parent R01 award. In the parent award, we are using iPSC technology to identify mechanisms of genetic cardiomyopathy in vitro and dissecting the role of crosstalk between cardiovascular cell types in pathogenesis. Mechanism underlying Down syndrome-related CHD involves complex intercellular communication leading to developmental and structural anomalies. Hence, we are confident that a comparative in vitro and bioinformatics analysis utilizing both Down syndrome and non-Down syndrome CHD iPSC-derived cardiomyocytes will likely extend our understanding of CHD as well as facilitate the discovery of novel genes and pathways that may be critical in its pathogenesis.

PUBLIC HEALTH RELEVANCE: Dilated cardiomyopathy (DCM) is among the most prevalent cause of inherited heart diseases. This study will investigate the molecular mechanisms of DCM by examining how proteins secreted from diseased cardiomyocyte can mediate crosstalk signaling to other cardiac cell types, including endothelial cells and fibroblasts. Results obtained here may provide insights into the pathogenesis of DCM and promote the development of therapeutics to rescue inter-cellular disease signaling pathways. Down syndrome (DS) is a common cause of congenital heart disease (CHD), yet its mechanism is poorly understood due in part to lack of suitable experimental models that can recapitulate the complexity of genotype-phenotype relationship. By using human induced pluripotent stem cells (iPSC), next generation sequencing (NGS), and bioinformatics strategy, we propose to examine the disruption of cellular communication signals in the heart with DS-related CHD. The results obtained here may provide us with important clues for pathogenesis of DS-related CHD as well as the development of disease-specific intervention strategies.